Requirement of GrgA for Chlamydia infectious progeny production, optimal growth, and efficient plasmid maintenance

ABSTRACT Chlamydia, an obligate intracellular bacterial pathogen, has a unique developmental cycle involving the differentiation of invading elementary bodies (EBs) to noninfectious reticulate bodies (RBs), replication of RBs, and redifferentiation of RBs into progeny EBs. Progression of this cycle is regulated by three sigma factors, which direct the RNA polymerase to their respective target gene promoters. We hypothesized that the Chlamydia-specific transcriptional regulator GrgA, previously shown to activate σ66 and σ28, plays an essential role in chlamydial development and growth. To test this hypothesis, we applied a novel genetic tool known as dependence on plasmid-mediated expression to create Chlamydia trachomatis with conditional GrgA deficiency. We show that GrgA-deficient C. trachomatis RBs have a growth rate that is approximately half of the normal rate and fail to transition into progeny EBs. In addition, GrgA-deficient C. trachomatis fails to maintain its virulence plasmid. Results of RNA-Seq analysis indicate that GrgA promotes RB growth by optimizing tRNA synthesis and expression of nutrient-acquisition genes, while it enables RB-to-EB conversion by facilitating the expression of a histone and outer membrane proteins required for EB morphogenesis. GrgA also regulates numerous other late genes required for host cell exit and subsequent EB invasion into host cells. Importantly, GrgA stimulates the expression of σ54, the third and last sigma factor, and its activator, AtoC, and thereby indirectly upregulating the expression of σ54-dependent genes. In conclusion, our work demonstrates that GrgA is a master transcriptional regulator in Chlamydia and plays multiple essential roles in chlamydial pathogenicity. IMPORTANCE Hallmarks of the developmental cycle of the obligate intracellular pathogenic bacterium Chlamydia are the primary differentiation of the infectious elementary body (EB) into the proliferative reticulate body (RB) and the secondary differentiation of RBs back into EBs. The mechanisms regulating these transitions remain unclear. In this report, we developed an effective novel strategy termed dependence on plasmid-mediated expression (DOPE) that allows for the knockdown of essential genes in Chlamydia. We demonstrate that GrgA, a Chlamydia-specific transcription factor, is essential for the secondary differentiation and optimal growth of RBs. We also show that GrgA, a chromosome-encoded regulatory protein, controls the maintenance of the chlamydial virulence plasmid. Transcriptomic analysis further indicates that GrgA functions as a critical regulator of all three sigma factors that recognize different promoter sets at developmental stages. The DOPE strategy outlined here should provide a valuable tool for future studies examining chlamydial growth, development, and pathogenicity.

C hlamydia is an obligate intracellular bacterium possessing a unique developmental cycle (1).The cycle involves two morphologically and functionally distinct cell types known as the elementary body (EB) and reticulate body (RB).The EB, approximately 0.3 µm in diameter, has DNA condensed by histones (2,3) and an outer membrane containing proteins crosslinked with disulfides (4).It is capable of temporary survival in extracellular environments despite having limited metabolic activities and is responsible for infecting host cells to initiate the developmental cycle.After EB is inside a host cell, its cysteine-rich outer membrane proteins undergo reduction, and DNA decondensation occurs.These processes enable EBs to differentiate into larger (approximately 1 µm in diameter) RBs that replicate within a cytoplasmic vacuole known as an inclusion.After multiple rounds of replication, RBs asynchronously differentiate back into non-replicat ing EBs (1,5).The newly formed EBs, upon release from host cells, can either infect other cells within the same host or transfer to new hosts.Unlike EBs, any released RBs are unable to initiate new developmental cycles.
The chlamydial developmental cycle is transcriptionally regulated (1,6,7).After EBs enter host cells, early genes are activated during the first few hours enabling primary differentiation into RBs.Starting at around 8 h post-infection, midcycle genes, represent ing the vast majority of all chlamydial genes, are expressed enabling RB replication.At around 24 h post-infection, late genes are activated to enable the secondary differentiation of RBs back into EBs.
Sigma factor is a subunit of the RNA polymerase (RNAP) holoenzyme that recognizes and binds specific DNA gene promoter elements, allowing RNAP to initiate transcription (8).Chlamydia encodes three different sigma factors termed σ66, σ28, and σ54 (9,10).σ66 RNAP holoenzyme is active throughout the developmental cycle, whereas the σ28 and σ54 RNAP holoenzymes transcribe only a subset of late or mid-late genes (11)(12)(13).
GrgA is a Chlamydia-specific transcriptional regulator that binds both to σ66 and σ28 and activates the transcription of numerous chlamydial genes in vitro and in vivo (14)(15)(16).RNA-Seq analysis of Chlamydia trachomatis conditionally overexpressing GrgA, along with GrgA in vitro transcription assays, revealed two other transcription factorencoding genes, euo and hrcA, as members of the GrgA regulon (14).Both euo and hrcA are transcribed during the early phase and midcycle (14).Euo acts as a repressor of chlamydial late genes (17), while HrcA regulates the expression of multiple protein chaperones (18), crucial for bacterial growth (19).These findings suggest GrgA plays an important regulatory role in chlamydial development.
Despite recent advancements in the genetic manipulation of Chlamydia (20,21), the tools available to study essential genes in this obligate intracellular bacterium remain limited.We attempted but failed to disrupt grgA through group II intron (Targetron) insertional mutagenesis (22).Previously, the Valdivia group was also unable to generate grgA-null mutants using chemical mutagenesis (23).Given that Targetron and chemical mutagenesis have been successfully used to disrupt numerous non-essential chlamydial genes [e.g., (24)(25)(26)(27)(28)(29)(30)], these negative results suggest that grgA is an essential gene for Chlamydia viability.In this work, we confirm that grgA is indeed an essential gene by using a novel genetic tool that we term DOPE (dependence on plasmid-mediated expression).Importantly, we show that GrgA is necessary for RB-to-EB differentiation and is also required for optimal RB growth.We further demonstrate that GrgA regulates the maintenance of the normal copy number of the plasmid, which encodes Pgp3 (a secreted virulence factor), Pgp4 (a transcriptional regulator of specific chromosomal genes as well as Pgp3), and proteins involved in plasmid replication (31)(32)(33).These findings provide further evidence that GrgA is a major regulator of σ66, σ28, and σ54 target genes, and plays important regulatory roles in governing the chlamydial developmental cycle.

DOPE enables grgA disruption
Targetron is a group II intron-based insertional mutagenesis technology that has been used successfully to disrupt numerous chlamydial chromosomal genes (24)(25)(26)(27)(28)(29)(30).In an effort to knock out GrgA expression in Chlamydia and investigate its physiological functions, we utilized Targetron vectors containing spectinomycin-resistance gene-bear ing group II introns specific for multiple grgA insertion sites.Whereas we did obtain spectinomycin-resistant chlamydiae, diagnostic PCR analysis detected the intron but not within grgA.Taken together with the earlier unsuccessful attempts to generate grgA-null mutants using chemical mutagenesis (23), we hypothesized that grgA is an essential gene and, as such, not amenable to conventional mutagenesis approaches.
To circumvent this issue, we devised a strategy we term DOPE to investigate the biological functions of essential genes in Chlamydia.Although disruption of essen tial genes in the wild-type bacterium causes lethality, transforming Chlamydia with a recombinant plasmid carrying the essential chromosomal gene downstream of an inducible promoter allows for the disruption of the chromosomal allele when the inducer is present in the culture medium.This method generates a strain with a disrupted essential gene, where withdrawal of the inducer will cause depletion of the gene products from the recombinant plasmid, allowing functional and mechanistic analyses of the essential gene.
We applied DOPE to study GrgA by constructing a shuttle vector named pGrgA-DOPE, which encodes an anhydrotetracycline (ATC)-inducible grgA allele (plasmid-encoded inducible grgA or peig) (Fig. S1).In addition to the replication origin, we kept all eight genes encoded by the wild-type C. trachomatis plasmid in the shuttle vector.This is because Pgp1, 2, 6, and 8 are essential for plasmid maintenance, while Pgp3 and 4 are a virulence determinant and regulator of certain chromosomal genes, respectively (31)(32)(33).Compared to the native chromosomal grgA allele containing a Targetron-insertion site between nucleotides 67 and 68 (Fig. 1A top), the grgA allele in pGrgA-DOPE carries a His-tag sequence and four synonymous point mutations around the intron-targeting site (Fig. 1A middle), rendering peig resistant to the Targetron designed for this site.Since ATC-induced GrgA overexpression previously caused C. trachomatis growth inhibition (14,34), we employed a weakened ribosomal binding site identified via a green fluorescence protein reporter (Fig. S2A through D) in pGrgA-DOPE to drive the expres sion of His-tagged GrgA (His-GrgA).We also removed a region that contained potential alternative −35 and −10 promoter elements between the ATC-inducible promoter and the ribosomal binding site from pGrgA-DOPE (Fig. S2E).
We transformed wild-type C. trachomatis L2 bearing an intact chromosomal grgA (i.e., L2/cg) with pGrgA-DOPE to derive L2/cg-peig.Western blotting demonstrated compara ble amounts of His-GrgA and endogenous GrgA at 12 h postinoculation (hpi) in L2/cgpeig cultures containing 0 to 5 nM ATC (Fig. S3A).We also analyzed the growth of L2/cgpeig in the presence of 0 or 1 nM ATC and found that ATC-induced GrgA overexpression did not affect the expression level of mKate2 [a red fluorescence protein encoded by pGrgA-DOPE (Fig. S1)], the inclusion size, chlamydial chromosome replication kinetics, or progeny EB production (Fig. S3B through F).These findings indicate that we can induce recombinant GrgA expression from pGrgA-DOPE at normal physiologic levels without adverse effects, thereby increasing the likelihood that we will be able to compensate for the loss of endogenous GrgA following the experimental disruption of the endogenous chromosomal grgA allele.

GrgA-deficient chlamydiae display slower RB growth and fail to form progeny EBs
To examine the impact of GrgA deficiency on C. trachomatis growth and development, we compared the growth of L2/cgad-peig in media with and without 1 nM ATC.Interestingly, immunofluorescence imaging using a major outer membrane protein (MOMP)-specific antibody revealed slower growth in ATC-free cultures as indicated by significantly smaller inclusions (Fig. 2A).Quantitative PCR analysis further confirmed the slower growth in GrgA-deficient cultures as revealed by slower chromosome replication in the absence of ATC (Fig. 2B; Fig. S3).The chromosome doubling time of L2/cgadpeig in the presence of ATC was 2 h (Fig. 2B), identical to the previously reported doubling time of wild-type L2 (14).In contrast, the doubling time of L2/cgad-peig in the absence of ATC doubled to 4 h (Fig. 2B).Intriguingly, ATC-free cultures exhibited a drastic reduction in the production of infectious progeny EBs (Fig. 2C).On average, each ATC-containing culture produced >10 5 inclusion-forming units (IFUs) at 24 hpi, near 10 7 at 34 hpi, and >10 8 at 48 and 72 hpi (Fig. 2C).By stark contrast, ATC-free cultures produced no detectable IFUs at 24 hpi and only about 300 s EBs at 34 h and thereafter (Fig. 2C).The severe decrease in EB production occurred despite chlamydial chromosome copy number in the ATC-free cultures at 34 hpi being equivalent to that in the ATC-containing cultures at 24 hpi (Fig. 2B).Consistent with the EB quantification assays, ultra-thin section transmission electron microscopy (EM) readily detected EBs and intermediate bodies (IBs) at 35 and 45 hpi in the ATC-containing cultures, whereas EBs were undetectable and IBs were nearly undetectable at 35, 45, or 60 hpi (Fig. 3A) in the ATC-free cultures.Collectively, these results demonstrate a requirement for GrgA for optimal RB growth and the production of EBs.

tetR mutations enable GrgA expression and EBs to escape in the absence of ATC
Although ATC-free L2/cgad-peig cultures exhibited a near complete deficiency in EB formation, we were still able to detect a very low background level of EB production (Fig. 2C).Unlike parental L2/cgad-peig, EBs collected from the ATC-free cultures (i.e., eL2/ cgad-peig) grew in subsequent ATC-free cultures as efficiently as in ATC-containing cultures, as evidenced by the morphology of immunostained inclusions (Fig. 4A) and results of IFU assays (Fig. 4B).Based on the inheritable ATC-independent growth exhibited by the escaped EBs, we hypothesized that spontaneous mutations in the tetR gene and/or tetO (TetR operator) might impair the grgA repression in L2/cgad-peig, thereby allowing for ATC-independent GrgA expression and the production of low levels L2/cg, mutations surrounding this site conferring resistance to intron targeting in peig, and grgA-intron joint regions in the chromosome of L2/cg-peig.Wild-type bases and corresponding mutated bases are shown with arrowheads and asterisks, respectively.(D) Western blotting showing time-dependent loss of His-GrgA in L2/cgad-peig upon ATC withdrawal.HeLa cells infected with L2/cgad-peig were cultured in the presence of 1 nM ATC.Three cultures were switched to ATC-free medium at 15 h postinoculation hpi.
Cultures were harvested with SDS-PAGE sample buffer at indicated times and resolved by SDS-PAGE.The membrane was first probed with mouse monoclonal MC22 anti-major outer membrane protein (MOMP) antibody, striped, and then reprobed with a polyclonal rabbit anti-GrgA antibody.
of EBs to form in the absence of ATC (Fig. 4C).To test this hypothesis, we first performed western blotting for cells infected with L2/cgad-peig or eL2/cgad-peig.These cells were exposed to ATC-containing medium from 0 to 18 hpi or initially for 16 h, then switched to ATC-free medium for the final 2 h.Notably, ATC withdrawal led to GrgA depletion in L2/ cgad-peig but did not alter GrgA expression in eL2/cgad-peig.We further recovered pGrgA-DOPE plasmids from EBs formed in ATC-free cultures.Significantly, DNA sequenc ing revealed a single nucleotide polymorphism (SNP) in the tetR gene in each of the 10 plasmids analyzed.Two of these SNPs lead to premature termination at codons 16 or 158, while the third SNP induces a frameshift at codon 64 (Fig. 4E).These observations support our hypothesis that lack of an authentic TetR results in the unchecked expres sion of wild-type GrgA in the absence of ATC (Fig. 4C).Together with findings in Fig. 3 and  4, these data further substantiate our supposition that GrgA is necessary for EB produc tion.

GrgA-deficient chlamydiae fail to maintain the virulence plasmid
Fluorescent proteins such as mKate2 are often employed as convenient markers for tracking genetic transformants (35,36).As depicted in Fig. S3B, L2/cg-peig produced mKate2-positive inclusions in both ATC-containing and ATC-free media.When cultured in ATC-containing medium, L2/cgad-peig also displayed mKate2-positive inclusions.However, when cultured in ATC-free medium, the usual mKate2-positive inclusions were largely absent, with only sporadic mKate2 signals observed, as elaborated below.
To analyze these phenomena under higher resolution, we metabolically labeled chlamydiae with a green-fluorescing lipid (N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]) aminocaproylsphingosine (C6-NBD-ceramide).The metabolic lipid labeling method is based on the host cell's ability to uptake the lipid during the pulse phase and transfer it to the chlamydiae during the chase phase (37,38).This enables the simultaneous visualization of chlamydiae (depicted in green) and the recombinant plasmid marker mKate (in red) in live cultures using fluorescence microscopy.Because L2/cgad-peig cultures exhibit slower growth in the absence of ATC, and that the number of chromo somes at 24 hpi in ATC-containing cultures is roughly equal to those at 34 hpi in ATC-free cultures (Fig. 2A and B), metabolic lipid labeling was initiated at 17.5 and 27.5 hpi for ATCcontaining and ATC-free cultures, respectively, and fluorescence microscopy was conducted at 24 and 34 hpi, respectively.
In ATC-containing but ampicillin-free cultures, almost 100% of inclusions exhibited strong colocalization of green fluorescence lipids and mKate2 signals, as demonstrated by significant color shifts following the merging of the lipid and mKate2 signals (Fig. 5A and B).This indicates widespread plasmid maintenance in chlamydiae.qPCR analysis detected 2.9 to 5.8 plasmids per chromosome (i.e., per cell) during the developmental cycle in ATC-enriched cultures, a count comparable to the native plasmid copy number (4.0 to 7.6 plasmids per chromosome) previously reported for wild-type C. trachomatis (39) (Fig. 5C).These findings suggest that L2/cgad-peig, with plasmid-expressed GrgA, is capable of maintaining its virulent plasmid even in the absence of the recombinant plasmid selection agent ampicillin (Fig. S1).
By contrast, in ATC-free cultures, no inclusions exhibiting diffused mKate2 signals of sufficient intensity to allow for significant color shifts upon merging with chlamydial lipid signals were detected (Fig. 5A and B).Indeed, the plasmid per chromosome ratio dropped to 0.5 at 24 hpi, gradually rose to 1.2 by 48 hpi, and remained at this level at 72 hpi (Fig. 5C).These observations imply that GrgA-deficient chlamydiae fail to maintain plasmid copies.Although inclusions in ATC-free cultures do not express diffused mKate2 signals, approximately 20% of the inclusions contained strong, punctate mKate2 signals, hinting at a potential plasmid segregation defect.Notably, ampicillin failed to enhance mKate2 expression (Fig. 5D and E) and only a ~2-fold increase in the plasmid copy number in ATC-free cultures (Fig. 5F), suggesting that the antibiotic that typically selects for the recombinant plasmid under "normal" conditions is unable to maintain the plasmid in the absence of GrgA.

GrgA deficiency leads to insufficient late gene activation
Given that GrgA is a transcriptional activator and that GrgA-deficient RBs fail to differentiate into EBs, we hypothesized that presumptive GrgA regulatory target genes crucial for EB formation may not be adequately activated in GrgA-deficient RBs.To identify these regulatory target genes, we performed RNA-Seq analysis on L2/cgad-peig cultured with and without ATC.We conducted RNA-Seq for ATC-containing cultures at 18 hpi and 24 hpi, corresponding to the midcycle point and early late developmental cycle point, respectively, in L2/cgad-peig (Fig. 2B and C), as well as wild-type C. trachomatis (6,14,34).Considering the slower growth rate of L2/cgad-peig cultured in ATC-free medium (Fig. 3), we extended the culture time to 24 hpi and 36 hpi before extracting RNA.qPCR analysis confirmed that chromosome copy numbers in ATC-containing cultures at 18 hpi were the same as those in ATC-free cultures at 24 hpi, while chromosome copy numbers of ATCcontaining cultures at 24 hpi were comparable to those of ATC-free cultures at 36 hpi (Fig. 6A).
We obtained on average of 6.3 million RNA-Seq reads mapping to the C. trachomatis genome per sample, representing 600 times genome coverages.Principal component analysis revealed strong consistencies within biological triplicates but notable differences among groups based on culture conditions and developmental stages (Fig. 6B).Consistent with the GrgA protein expression data in Fig. 1D and plasmid copy number data (Fig. 5C), RNA-Seq detected 204-and 157-fold decreases in the transcripts of his-grgA encoded by pGrgA-DOPE in the ATC-free cultures of L2/cgad-peig at the corre sponding midcycle and early late developmental points, respectively, compared with ATC-containing cultures (Table S1).
We identified 150 genes activated by 2-to 56-fold (P ≤0.05) in ATC-containing cultures from 18 hpi (midcycle) to 24 hpi (early late stage) (Table S2).Most likely, these late genes either drive or are consequent to the conversion of RBs into EBs.Indeed, the top two ranked genes encode an EB-enriched cysteine-rich outer membrane protein OmcA and a histone HctB, both essential for EB morphogenesis.When changes of these 155 late genes in the ATC-free cultures from 24 hpi (midcycle) to 36 hpi (early late stage) were plotted alongside the ATC-containing cultures (Table S2), it was notable that 43 of the 45 genes with ≥5-fold expression increases in the ATC-containing cultures failed to increase to the same degree in the ATC-free cultures (Fig. 6C; Table S2).For example, hctB increased 41.2-fold in ATC-containing cultures but only 5.3-fold in ATC-free cultures.omcA increased 56.2-fold in ATC-containing cultures but only 21.1-fold in ATC-free cultures (Fig. 6C; Table S2).Similarly, omcB increased 20.5-fold in ATC-containing cultures but only 4.9-fold in ATC-free cultures (Fig. 6C; Table S2).The protease-encoding gene tsp, thought to degrade certain RB-specific proteins toward the end of midcycle (40), exhibited a 5.5-fold increase in ATC-containing cultures but only a 1.7-fold increase in ATC-free cultures (Fig. 6C; Table S2).Results of quantitative reverse transcription-PCR (qRT-PCR) analysis demonstrated consistent increases in omcA, omcB, hctB, and tsp in ATC-containing cultures but not in ATC-free cultures from midcycle to early late cycle (Fig. 6D).These findings support the notion that GrgA is critical for adequate activation of late genes involved in the RB-to-EB conversion.
RNA-Seq detected a higher hctA expression increase in ATC-free cultures than in ATCcontaining cultures from the midcycle to the early late developmental point, compared to a reduced hctB expression increase (Fig. 6C; Table S2).However, qRT-PCR analysis revealed similar hctA expression increases in ATC-free and ATC-containing cultures between the two developmental points (data not shown).This suggests that GrgA does not regulate hctA expression.

GrgA deficiency downregulates expression of target genes of σ28 and σ54
Our previous in vitro study showed that GrgA can directly activate transcription from σ28-dependent promoters in addition to σ66-dependent promoters (15).Notably, the aforementioned hctB and tsp downregulated in ATC-free cultures of L2/cgad-peig at early late developmental points constitute the entire σ28 regulon (46).This in vivo observation confirms that GrgA regulates the expression of σ28 genes in Chlamydia.
Two research groups have proposed partially overlapping gene sets as the σ54 regulon in C. trachomatis.While Soules et al. suggested 64 targets through overexpres sion of the σ54 activator atoC (ctcC) (11), Hatch et al. proposed nearly 30 targets through σ54 depletion (46).Significantly, of the 64 genes exhibiting ≥2-fold decreases in ATC-free cultures, 42 genes, including the aforementioned omcA, omcB, hctB, and numerous T3SSrelated genes, are σ54 targets proposed by either or both studies (Table 1).These observations suggest that GrgA also regulates the expression of σ54 genes.
We noticed moderate (about 1.5-fold) yet significant decreases in transcripts of rpoN (σ54), atoC (ctcC), and atoS (ctcB, a sensor kinase gene cotranscribed with atoC) in ATC-free cultures of L2/cgad-peig at the early late developmental point.We performed qRT-PCR analysis and confirmed that all these three genes showed significant ≥2-fold decreases (Fig. 7).These observations suggest that GrgA regulates the expression of σ54 genes by controlling the expression levels of σ54 and its regulators of atoC (ctcC) and atoS (ctcB).

GrgA deficiency disrupts the midcycle transcriptome
Our discovery of early late stage transcriptomic changes underlying EB formation deficiency prompted us to examine midcycle transcriptomic differences in L2/cgad-peig cultures with and without ATC, to further discern the mechanisms by which GrgA regulates RB growth.A total of 28 chromosomal genes exhibited a 2.0-to 4.1-fold downregulation (P < 0.05) in ATC-free L2/cgad-peig cultures as determined by RNA-Seq.Interestingly, of these 28 chromosomal genes, 7 (25%) encode tRNAs (Table 2).Of note, 10 other tRNA genes displayed 1.5-to 2.0-fold decreases (P < 0.05, Table S1).Together, these 17 tRNAs constitute nearly half of the 37 tRNAs expressed in C. trachomatis, thus suggesting that GrgA supports RB growth in part, by boosting tRNA expression and protein synthesis.Among the remaining 21 chromosomal genes with ≥2.0-fold expression decreases in ATC-free L2/cgad-peig cultures during the midcycle, oppD and oppF encode oligopeptide ABC transporter ATP-binding proteins, while ctl0674 encodes a metal ABC transporter, and trpB encodes tryptophan synthase B. These changes suggest that GrgA promotes RB growth by upregulating both nutrient acquisition from host cells and de novo tryptophan biosynthesis, which are both important for chlamydial growth (47,48).
Two additional genes of interest that are downregulated in ATC-free cultures of L2/ cgad-peig during the midcycle are obgE and nth, which encode DNA topoisomerase IV subunit B and endonuclease III, and are required for DNA replication and repair, respectively.These data suggest that GrgA plays a potential role in the regulation of DNA replication and repair during RB growth.
Interestingly, 70 chromosomal genes showed 2.0-to 5.3-fold increases in ATC-free cultures at the midcycle (P < 0.05) compared with ATC-containing cultures (Table S4).The number of upregulated genes is 2.5-fold higher than the number of downregula ted genes discussed above (Table 1).This finding raises the possibility that GrgA also functions to regulate the silencing of large networks of developmental genes.Most notably, two well-characterized transcription factors, euo (late gene repressor) and hrcA (heat-inducible transcriptional repressor), demonstrated aberrantly increased expression in the ATC-free cultures during the midcycle (Table S4).The increased expression of euo and hrcA was surprising because previous studies found that GrgA overexpression also increased euo and hrcA expression (14).qRT-PCR analysis confirmed that both euo and hrcA were indeed increased in ATC-free cultures of L2/cgad-peig (Fig. S4).These findings strengthen the notion that GrgA serves as a master transcriptional regulator in Chlamydia.
Although RB replication is apparently inhibited in the absence of GrgA, we observed that four genes involved in DNA replication and repair showed increased expression in ATC-free cultures of L2/cgad-peig during the midcycle time point.These include ruvB (Holliday junction DNA helicase RuvB), recA (recombinase A), ihfA (DNA-binding protein Hu), and ssb (single-stranded DNA-binding protein).Intriguingly, all four genes are also upregulated when chlamydial growth is halted in response to heat shock (49), thus suggesting a potential regulatory role for GrgA in modulating environmental stress responses in Chlamydia.Similarly, four protease genes (lon, htrA, ftsH, and ctl0301), four protein chaperone genes (clpB, clpC, groEL, and groES), and two enzymes that regulate disulfide bonding in proteins (ctl0152 and trxB) also displayed increased expression in ATC-free cultures during the midcycle.In addition to the aforementioned tRNA downregulation, the upregulated protease, chaperone, and disulfide isomerase genes likely contribute to protein homeostatic imbalance and functional impairment, resulting in RB growth inhibition.Taken together, midcycle RNA-Seq data suggest that GrgA promotes RB growth by optimizing the expression of tRNAs and certain nutrient transports.Furthermore, GrgA may function as both a transcriptional activator and a transcriptional repressor, as discussed below.

Pgp4 target genes are downregulated in GrgA-deficient chlamydiae at the early late developmental point
Through microarray analysis of wild-type C. trachomatis as well as variants without a recombinant plasmid or with a plasmid either carrying or not carrying pgp4 at 24 hpi, Song et al. identified Pgp4 as a positive transcriptional regulator of nine chromosomal genes (32).At the early late developmental stage, GrgA deficiency had effects on the expression of these chromosomal genes similar to plasmid and Pgp4 deficiencies (Table S5), suggesting that GrgA regulates these genes primarily through maintaining the virulence plasmid.However, this cannot be said for the midcycle developmental stage, as the effects of plasmid and Pgp4 deficiencies on the transcriptome have not yet been studied at that point.

DOPE as a valuable tool for study essential gene in Chlamydia and other obligate intracellular organisms
Since the first demonstration of reproducible Chlamydia transformation using a shuttle vector 12 years ago (35), the research community has leveraged this reverse genetic tool to investigate gene function via ectopic overexpression, insertional mutagenesis, deletion, and other methods (11,(24)(25)(26)(27)(28)(29)(30)(50)(51)(52)(53)(54).Nonetheless, the lack of effective strategies to disrupt truly essential genes, particularly those whose overexpression is toxic, has hampered research in Chlamydia and other biological systems.In this study, we developed a novel, tightly regulated, inducible expression system termed DOPE, which shares similarity with a system recently reported by Cortina et al (53).DOPE facilitates the functional examination of an essential gene by permanently disrupting the gene in the chromosome while conditionally depleting the gene products expressed by the complementing plasmid.
The DOPE system represents a convenient and versatile tool for establishing the essentiality of a gene and, at the same time, investigating the gene's underlying functional mechanisms.An advantage of DOPE over recently developed conditional CRISPR interference systems (51,55) is that gene depletion in DOPE is achieved by omitting ATC, whereas CRISPR interference relies on ATC's presence.The adverse effects of ATC on Chlamydia and other bacteria have been mitigated but not entirely eradica ted.In fact, it has been documented that chlamydial growth is inhibited with ATC at concentrations ≥20 nM (36).This makes the application of CRISPR interference to chlamydial infection in animal models challenging, as ATC concentrations in tissues and organs are not easily regulated.Furthermore, potential concerns arise about the adverse effects of ATC on the microbiota, as studies have shown that gut microbiota influences chlamydial pathogenesis (56).In addition to ATC independence, chromosomal gene mutants established using DOPE are devoid of potential off-target effects that may occur with CRISPR interference (57).

GrgA as one of the most important regulators of chlamydial physiology
GrgA is an exclusive Chlamydia-specific protein with no homologs in non-chlamydial organisms, yet highly conserved among chlamydiae, including environmental chlamy diae (16).By employing the DOPE strategy, we demonstrate that GrgA plays a critical role in sustaining RB replication efficiency and is absolutely essential for RB-to-EB differentiation (Fig. 3 and 4).
RNA-Seq analysis revealed numerous mechanisms through which GrgA regulates RB growth and EB formation.Perhaps chief among these regulatory mechanisms, during the midcycle, GrgA-deficient RBs exhibited decreased expression of numerous tRNA genes and ABC transporter genes, while displaying increased transcripts of protease genes and chaperone genes (Table S4).These findings suggest that GrgA enables optimal RB growth in part by facilitating RB protein synthesis and nutrient acquisition and by modulating post-translational protein homeostasis.Many other gene expression changes induced by GrgA deficiency (Table 1, Table S1) likely exert additive or syner gistic effects on RB growth.In addition to regulating chromosomal gene expression, our findings here show that GrgA may also regulate RB growth by enabling plasmid replication and segregation (Fig. 5).Consistent with this notion, plasmid-free Chlamydia muridarum has slower RB replication kinetics (58).Mechanistically, even though Pgp4 has been shown to function as a transcription regulator of chromosomal genes (Table S5) (32,59), its role in the transcriptomic expression during the midcycle has yet to be investigated.
The morphological signature features of EBs are their small size and high electron density.The RB-to-EB conversion necessitates DNA condensation and reorganization of the chlamydial envelope.These processes involve histones (i.e., HctA and HctB) and outer membrane proteins (e.g., OmcA and OmcB) (2-4), respectively.Significant in this regard, our transcriptomic analysis of the early late developmental stage suggests that GrgA facilitates RB-to-EB differentiation by activating the expression of the histone gene hctB and late-stage outer membrane protein genes omcA and omcB.Moreover, GrgA may aid EB formation by inducing the protease gene tsp and modulating numerous other genes (Fig. 6C and D; Tables S1 and S2).
Promoter reporter analysis suggests that hctA expression precedes hctB (60).Our transcriptomic analysis indicates that GrgA is required for increased expression of hctB but not hctA at the early late developmental point (Fig. 6C and D; Tables S2 and S3).Despite this, GrgA-deficient RBs fail to produce IBs, a transition cell type during the RB-to-EB conversion (Fig. 3).This finding implies that DNA condensation requires both the actions of both HctA and HctB.
In conjunction with optimizing RB growth and enabling EB formation, our RNA-Seq analysis suggests that GrgA plays additional important roles in the chlamydial develop mental cycle.For example, numerous genes encoding T3SS structure proteins, effectors, and chaperones were observed to be downregulated in GrgA-deficient chlamydiae.Among the downregulated genes were the effectors CTL0480 that interacts with the host myosin phosphatase pathway and regulates chlamydial exit (30,42), and TARP which is secreted from EBs immediately after they are taken up by host cells and interacts with host cytoskeleton protein actin to facilitate cell entry (41,43,44).Decreased transcripts of ctl0480 and tarp in GrgA-deficient chlamydiae were observed at the early late developmental stage, thus suggesting that GrgA helps facilitate EB exit from infected cells and invasion of new host cells, both of which are required for chlamydial dissemina tion.

GrgA as a crucial regulator of chlamydial plasmid maintenance
The plasmids of C. trachomatis and C. muridarum serve as important virulence factors in these two species (31,(61)(62)(63).Our results of lipid metabolic labeling and mKate imaging (Fig. 5) indicate that GrgA plays a crucial role in C. trachomatis plasmid replication and/or segregation.The plasmid loss is consistent with an apparent decrease in glycogen particles observed with EM in ATC-free cultures of L2/cgad-peig (Fig. 3A) since Pgp4 is required for efficient glgA (glycogen synthesis) expression (Table S5) (32).To the best of our knowledge, other than the standard DNA replication machinery components, GrgA is the first chromosome-encoded regulatory protein required for maintaining the chlamydial plasmid.
The mechanism through which GrgA maintains the virulence plasmid is unclear.Previous studies established that pgp1, pgp2, pgg6, and pgp8 are essential for the maintenance of the plasmid.Our qRT-PCR analysis and qPCR analyses demonstrated that the transcript/plasmid ratios for all pgp genes are actually higher in ATC-free cultures than in ATC-containing cultures (data not shown).These findings suggest that plasmid loss in GrgA-deficient chlamydiae is not caused by decreased pgp expression.

GrgA as a master transcriptional regulator
As a component of the RNAP holoenzyme, sigma factor recognizes and binds to specific promoter sequences (8,64,65).Based on the temporal expression profiles of chlamydial sigma factors and recent studies, it is generally recognized that σ66 is the principal housekeeping sigma factor, whereas σ28 and σ54 regulate the expression of certain late genes regulating the differentiation of RBs into EBs (11,12,46,66).While controversies exist regarding the exact composition of the σ28 and σ54 regulons in C. trachomatis (11,12,46,66), numerous previously reported σ28 and σ54 target genes were found to be downregulated by GrgA deficiency (Table 1).These findings indicate that GrgA not only regulates σ66 genes, but also σ28 and σ54 genes.
Previous studies suggest that distinct GrgA domains directly bind to σ66 and σ28 to regulate the expression of their respective target genes (15, 16) (Fig. 8).Among the numerous σ66 target genes are rpoN (σ54), atoC (activator of σ54), and atoS (presumed positive regulator of AtoC).Findings from this study (Fig. 7) indicate that GrgA stimulates the expression of all these three genes to upregulate σ54 target genes (Fig. 8).
In vitro transcription analysis established that GrgA functions as a transcriptional activator (14)(15)(16).Surprisingly, euo and hrcA, whose promoter activities were stimulated by GrgA in vitro and whose transcripts were increased by GrgA overexpression, exhibited increased transcripts in GrgA-deficient chlamydiae (Tables S1 and 4; Fig. S4).This seemingly inconsistency raises the possibility that increased euo and hrcA expression in GrgA-deficient chlamydiae could be the result of indirect regulation.However, the fact that GrgA deficiency resulted in 2.5 times more increased genes (Table S4) than decreased genes (Table S3) during the midcycle raises the possibility that GrgA could function as both an activator and a repressor.It is worth noting that other bacterial transcription factors can both activate and repress genes (67)(68)(69).For example, the cyclic AMP receptor protein binds different region of an outer membrane protein promoter and activates transcription by directly interacting with RNAP but can also repress the transcription after recruiting a transcriptional corepressor (67).The transcription factor Fur can either inhibit RNAP binding to the promoters of iron-uptake genes or facilitate its recruitment to other gene promoters (70).We speculate that GrgA could regulate its target genes in an analogous manner.Alternatively, rather than being directly activated by GrgA, some σ66-dependent genes might exhibit enhanced expression as a result of increased availability of σ66-RNAP when GrgA is absent and not directing it to GrgA target genes.Taken together, GrgA regulates the expression of target genes of all three sigma factors, albeit through distinct mechanisms; GrgA also regulates the expression of other transcription factors.These functions validate GrgA's role as a master transcrip tional regulator in Chlamydia.
In summary, DOPE has enabled us to disrupt an essential chromosomal gene grgA.We show that GrgA serves as a checkpoint for chlamydial secondary differentiation and a crucial regulator of RB growth and plasmid maintenance.The formation of EBs is absolutely required for dissemination of chlamydial infection within the infected host and transmission to new hosts.Because RBs and EBs share most of the immunodominant antigens (e.g., major outer membrane protein), conditional GrgA-deficient, "maturation"defective chlamydiae are potential candidates for live attenuated Chlamydia vaccines, provided that strategies are in place to fully prevent EBs from escaping the gene expression regulatory system in DOPE plasmid.To the minimum, the maturationdefective chlamydiae will serve as a useful system for studying the roles of RBs in antichlamydial immunity.

Host cells and culture conditions
Mouse fibroblast L929 cells were used as the host cells for C. trachomatis transformation and preparation of highly purified EBs.Human vaginal carcinoma HeLa cells were used for experiments determining the effects of depletion on chlamydial growth and development.Both L929 and HeLa cell lines were maintained as monolayer cultures using Dulbecco's modified Eagle's medium (DMEM) (Sigma Millipore) containing 5% and 10% fetal bovine serum (vol/vol), respectively.Gentamicin (final concentration: 20 µg/mL) was used for maintenance of uninfected cells and was replaced with penicillin (10 units/mL) and/or spectinomycin (500 µg/mL) as detailed below.37°C, 5% CO 2 incubators were used for culturing uninfected and infected cells.

Plasmid extraction from eL2/cgad-peig
Cells in six-well plates were infected with EB to achieve ≥90% infection rate and cultured for 40 hpi.After removal of the medium, 200 µL H 2 O was added and cells were removed from the plates with the aid of a Cell Lifter (Corning).The lysate was collected into an Eppendorf tube containing 20 µL of 10× phosphate-buffered saline (PBS) to bring back isotonicity and centrifuged at 3,000 rpm using a Beckman desktop minifuge.The supernatant-containing chlamydial cells were collected and heated at 95°C for 5 min, centrifuged at 14,000 rpm in the minifuge.Plasmids released were purified with phenol/chloroform extraction and precipitated using ethanol.The precipitated DNA was dissolved in 5-10 µL H 2 O, which was used to transform E. coli.

Immunofluorescence staining
Because chlamydiae are obligate intracellular bacteria, traditional methods used for assessing free-living bacteria growth, such as optical density and agar plate colony-form ing unit measurements, are not suitable for determining chlamydial growth.Instead, immunostaining of C. trachomatis inclusions is used as a qualitative method to evaluate its growth.Near-confluent HeLa monolayers grown on six-well plates were inoculated with L2/cgad-peig at a multiplicity of infection of 0.3 inclusion-forming units per host cell.Following 20-min centrifugation at 900 g, cells were cultured at 37°C in media containing either 0 nM or 1 nM ATC for 30 h.The infected cells were then fixed with cold methanol, blocked with 10% fetal bovine serum prepared in PBS, and stained successively with the monoclonal L21-5 anti-major outer membrane protein antibody (73) and an FITC-conjugated rabbit anti-mouse antibody cells.Immunostained cells were finally counter-stained with 0.01% Evans blue (in PBS).Red (Evan blue) and green (MOMP) fluorescence images were acquired on an Olympus IX51 fluorescence micro scope equipped with an Olympus monochrome ICC camera or an Infinity i8-3 CMOS monochrome camera.A constant exposure time for each channel was used for cultures in the same experiments.Overlay of images obtained with the Olympus camera and Infinity i8-3 camera was performed using the PictureFrame software and ACINST03 respectively.The Java-based ImageJ software was then used to calculate areas and mKate intensities of inclusions (34).

IFU assay
The IFU assay, which quantifies EBs by determining IFUs in host cells, serves essentially as a chlamydial colony-forming unit assay.Frozen purified L2/cgad-peig EB stock or crude harvests of L2/cgad-peig cultured with or without ATC were thawed, 1-to-10 serially diluted, and inoculated onto L929 monolayers grown on 96-well plates using medium containing 1 nM ATC and 1 µg/mL cycloheximide.Following 20-min centrifugation at 900 g, cells were cultured at 37°C for 30 h. Cell fixation and antibody reactions were performed as described above.Immunostained inclusions were counted under the fluorescence microscope without Evan blue counter-staining.

Diagnostic PCR and DNA sequencing
For confirming and sequencing grgA alleles in the chromosome and plasmid, total DNA was extracted from ~1,000 infected cells using the Quick-gDNA MiniPrep kit (Sigma Millipore) following manufacturer's instructions.The resulting DNA was used as template for PCR amplification using Taq DNA polymerase (Genscript).DNA fragments resolved with electrophoresis of 1.2% agarose gel purified using the Gel Extraction Kit (Qiagen) and subject to Sanger sequencing at Quintara Biosciences.

Quantification of chromosome and plasmid copy numbers
The chromosome copy number provides a quantitative measurement of the number of chlamydial cells, including RBs, EBs, and IBs, and is particularly useful for quantifying RB growth since RBs are non-infectious.To quantify chromosome copy numbers in cultures, infected cells were detached from 12-well plates using Cell Lifters (Corning).Cells and media were collected into Eppendorf tubes, centrifuged at 20,000 g at 4°C.The supernatant was carefully aspirated.One hundred microliters of alkaline lysis buffer (100 mM NaOH and 0.2 mM EDTA) was added into each tube to dissolve the cell pellets.Tubes were heated at 95°C for 15 min and then placed on ice.Three hundred fifty microliters of H 2 O and 50 µL of 200 mM Tri-HCl (pH 7.2) were added into each tube and mixed.The neutralized extracts were used for qPCR analysis (1 µL/reaction) directly for samples collected up to 24 hpi or after a 100-fold dilution for samples collected thereafter.A pair of ctl0631 primers (Table S5) was used for qPCR analysis to quantify chromosome copy numbers, while a pair of pgp1 primers (Table S5) was used to quantify plasmid copy numbers.qPCR analysis was performed with biological triplicates and technical duplicates using QuantStudio five real-time PCR System and Power SYBR Green PCR Master Mix (ThermoFisher Bioscientific) (14,34).
We took advantage of the pGrgA-DOPE plasmid to quantify the plasmid-to-chromo some ratio without preparing chlamydial chromosomal DNA free of host DNA contam ination.Briefly, a pair of grgA primers and the aforementioned pgp1 primers were simultaneously used to quantify pGrgA-DOPE prepared from Escherichia coli, while the grgA primers (Table S5) and the aforementioned ctl0631 primers were simultaneously used to quantify chromosome of wild-type C. trachomatis.These analyses showed that the grgA primers and pgp1 primers have the same amplification efficiencies for quantifying pGrgA-DOPE, while the grgA primers is 30% more efficient than the ctl0631 primers in amplifying the chromosome.To determine plasmid-to-chromosome ratio in L2/cgad-peig, we ran qPCR analysis for pgp1 and ctl0631 simultaneously.We calculated plasmid-to-chromosome ratios by correcting the ctl0631 amplification efficiency with grgA amplification efficiency.

Western blotting
Detection of MOMP and GrgA was performed as previously described (34).Chlamydiainfected cells in each well were harvested in 200 µL of 1× SDS-PAGE sample buffer, heated at 95°C for 5 min, and sonicated for 1 min (5 s on/5 s off) at 35% amplitude.Proteins were resolved in 10% SDS-PAGE gels and thereafter transferred onto poly vinylidene difluoride membranes.The membrane was probed with the monoclonal mouse anti-MOMP MC22 antibody (74), stripped, and reprobed with a polyclonal mouse anti-GrgA antibody (34).

Transmission electron microscopy
To visualize intracellular chlamydiae up to 36 hpi, L929 cell monolayers grown on six-well plates were infected as described above and cultured with medium supplemented with or without 1 nM ATC.For cultures up to 36 h, cells were removed from the plastic surface using trypsin, collected in PBS containing 10% fetal bovine serum, and centrifuged for 10 min at 500 g.Pelleted cells were resuspended in EM fixation buffer (2.5% glutaralde hyde, 4% paraformaldehyde, 0.1 M cacodylate buffer) at RT, allowed to incubate for 2 h, and stored at 4°C overnight.To visualize intracellular chlamydiae at 45 and 60 hpi, the above procedures resulted in lysis of infected cells and inclusions.To overcome this problem, cells grown on glass coverslips were infected with and fixed without trypsiniza tion.To prepare samples for imaging, cells were first rinsed in 0.1 M cacodylate buffer, dehydrated in a graded series of ethanol, and then embedded in Eponate 812 resin at 68°C overnight.Ninety-nanometer thin sections were cut on a Leica UC6 microtome and picked up on a copper grid.Grids were stained with uranyl acetate followed by lead citrate.TIFF images were acquired on a Philips CM12 electron microscope at 80 kV using an AMT XR111 digital camera.EBs, RBs, and IBs were enumerated.

RNA isolation
Total host and chlamydial RNA were isolated from L2/cgad-peig-infected HeLa cells using TRI reagent (Millipore Sigma).DNA decontamination was achieved by two cycles of DNase I-XT (New England Biolabs) digestion.Complete removal of genomic DNA was confirmed by PCR analysis.RNA concentration was determined using Qubit RNA assay kits (ThermoFisher).Aliquots of the DNA-free RNA samples were stored at −80°C.

RNA sequencing and analyses
RNA-Seq was performed as described with minor modifications (14,49).Briefly, total RNA integrity was determined using Fragment Analyzer (Agilent) prior to RNA-Seq library preparation.Illumina MRZE706 Ribo-Zero Gold Epidemiology rRNA Removal kit was used to remove mouse and chlamydial rRNAs.Oligo(dT) beads were used to remove mouse mRNA.RNA-Seq libraries were prepared using Illumina TruSeq stranded mRNA-Seq sample preparation protocol, subjected to quantification process, pooled for cBot amplification, and sequenced with Illumina Novoseq platform with 100 bp pair-end sequencing module.Short read sequences were first aligned to the CtL2 434/Bu including the chromosome (GCF_000068585.1_ASM6858v1),the pL2 plasmid (AM886278), and four genes cloned into the plasmid (his-grgA, bla, tetR and the mKate gene) using STAR version 2.7.5a and then quantified for gene expression by HTSeq to obtain raw read counts per gene, and then converted to FPKM (Fragment Per Kilobase of gene length per Million reads of the library) (75)(76)(77).DESeq2, an R package commonly used for analysis of data from RNA-Seq studies and test for differential expression (78), was used to normalize data and find group-pairwise differential gene expression based on three criteria: P < 0.05, average FPKM >1, and fold change ≥1.

Statistical analysis
For the evaluation of progeny EBs, plasmid-to-chromosome ratios, qPCR results, qRT-PCR results, inclusion areas, and inclusion mKate intensities, t-tests were conducted using Microsoft Office Excel.Where applicable, P-values were adjusted for multiple compari sons by Benjamini-Hochberg procedure to control the false discovery rate.

FIG 2
FIG 2 GrgA deficiency slows RB growth and disables the formation of infectious progeny.L2/cg-peig-infected HeLa cells were cultured in the presence or absence of 1 nM ATC.At indicated hpi, cultures were terminated for immunofluorescence assay (A), genome copy quantification (B), or quantification of inclusion-forming unit (C).(A) Infected cells were fixed with methanol, sequentially reacted with monoclonal mouse L2-5 anti-MOMP antibody and a fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG secondary antibody, counter-stained with Evan blue, and imaged using a fluorescence microscope.(B) Total genomic DNA, prepared as detailed in Materials and Methods, was used for C. trachomatis chromosome quantification by employing primers targeting ctl0631.(C) Infected cells were harvested in sucrose-phosphate-glutamate buffer, disrupted by sonication, and inoculated onto monolayers grown on 96-well plates following 10-fold serial dilution.Recoverable inclusion-forming units were detected by immunostaining as described in (A).(B, C) Data represent averages ± standard deviations of triplicate cultures.

FIG 3 FIG 4
FIG 3 Lack of EB formation in GrgA-deficent cultures.(A) Representative transmission electron microscopic (EM) images of L2/cgad-peig cultured in media containing either 0 nM or 1 nM at indicated hpi.ATC-containing culture was not processed for EM at 60 hpi because nearly all inclusions already burst by that point.Representative RBs, EBs, and IBs are marked by green, red, and orange arrows, respectively.Note that small irregularly shaped electron-dense particles with representative ones pointed to by black arrows, in both ATC-containing and ATC-free cultures, are glycogen particles.Size bar equals 2 µm.(B) Scattergraphs of RBs, EBs, and IBs counted from multiple inclusions.

FIG 5
FIG 5 GrgA deficiency causes plasmid loss.(A, B) L2/cgad-peig cultured with ampicillin-free media containing 0 nM or 1 nM ATC was metabolically labeled with the fluorescent lipid C6-NBD-ceramide as described in Materials and Methods.C6-NBD-ceramide-labeled Chlamydia (green) and mKate expressed from the plasmid are imaged under a fluorescence microscope.(A) Representative images of live cultures of L2/cgad-peig labeled with C6-NBD-ceramide.Note that the red fluorescence protein mKate2 is expressed by pGrgA-DOPE, which also encodes a β-lactamase rendering ampicillin resistance.(B) Scattergraph of percentages of inclusions with broad color shift after merging green and red channels from multiple images from cultures described in A. (C) Kinetics of plasmids per chromosome in L2/cgad-peig cultured with ampicillin-free media containing 0 nM or 1 nM ATC.Total DNA was prepared at indicated times.qPCR analysis was performed for the (Continued on next page)

FIG 5 (
FIG5 (Continued)    chromosomal gene ctl0631 and the plasmid gene pgp1.The plasmid/chromosome ratio was derived as described in Materials and Methods.Quantification of the pGrgA-DOPE and chromosome was carried out with qPCR as described in Materials and Methods.Data represent averages ± standard deviation from biological triplicates.(D, E) L2/cgad-peig cultured with 0 nM or 1 nM ATC plus 10 µg/mL ampicillin was metabolically labeled with the fluorescent lipid C6-NBD-ceramide.(D) Representative images of live cultures of L2/cgad-peig labeled with C6-NBD-ceramide with or without 1 nM ATC.(E) Scattergraph of percentages of inclusions with broad color shift after merging green and red channels from multiple images from cultures described in A. (F) Kinetics of plasmids per chromosome cultured with 0 nM and 1 nM ATC plus 10 µg/mL ampicillin as determined in panel C.

FIG 6
FIG 6 GrgA deficiency disrupts late gene activation.RNA and DNA were prepared from L2/cgad-heig and were cultured with 0 nM or 1 nM ATC.(A) Timing of RNA extraction for RNA-Seq analysis of ATC-containing and ATC-free cultures of L2/cgad-peig and equivalent chromosome copies in two types cultures at the defined midcycle points as well as the early late cycle points.(B) High intragroup consistency of RNA-Seq data revealed by principal component analysis.(C) Most of late genes with ≥5-fold increases in ATC-containing cultures had lower degree of increases in ATC-free cultures in RNA-Seq analysis.(D) Confirmation of insufficient activation of four late genes in ATC-free cultures by quantitative reverse transcription-PCR analysis.Data were averages ± standard deviations of biological triplicates.

FIG 7
FIG 7Confirmation of downregulated expression of rpoN and its regulators atoC and atoS.RNA from Fig.6was used for qRT-PCR analysis.Data were averages ± standard deviations of biological triplicates.

FIG 8
FIG 8Proposed mechanisms for regulation of σ66, σ28, and σ54 target genes by GrgA.Distinct regions of GrgA interact with σ66 and σ28 to directly regulate the transcription from their target gene promoters by the RNA polymerase core enzyme (comprised of α, β, β' , and ω subunits).Among σ66-dependent genes are rpoN (σ54) and atoC, which are upregulated by GrgA.Accordingly, σ54 target genes are indirectly upregulated by GrgA. Figure was generated using paid subscription to Biorender.

TABLE 1
σ54 targets downregulated by GrgA deficiency at early late developmental points a Abbreviation, DT and IT, direct targets and indirect targets, respectively, defined by Soules et al. through RNA-Seq analysis of atoC-overexpressing chlamydiae, promoter sequence analysis, and transcription report assays in (46)cherichia coli(11).T, targets defined by Hatch et al. through RNA-Seq analysis of σ54-depleted chlamydiae(46).